Antibacterial Activity Of Euphorbia Hirta (L.): Euphorbiaceae And Isolation Of Its Bioactive Constituents
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Date
2015-08
Authors
Perumal, Shanmugapriya
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Abstract
Natural products, especially medicinal plants, remain as a valuable source
offering a revolutionary green approach to the discovery of novel antibacterial
compounds. Euphorbia hirta (L.) plant is traditionally used in many tropical countries
for the treatment of gastrointestinal, bronchial and respiratory ailments caused by
nosocomial infectious agents. In this study, various solvent extracts (methanol,
ethanol, ethyl acetate, dichloromethane and hexane) of E. hirta were subjected to
phytochemical screening to determine total phenolic and flavonoid contents. The
potential antibacterial activities of the crude extracts were assessed via in vitro
antibacterial test using broth microdilution method against a panel of clinically
resistant pathogens and American Type Culture Collection (ATCC) strains. The most
bioactive extract, the methanol extract was further purified in a bioactivity-guided
fractionation and isolation method using column chromatography, which finally
yielded three pure antibacterial compounds. The chemical structures of these purified
compounds were characterized and identified through analyses of spectroscopic
methods. The isolated antibacterial constituents were investigated for the antibacterial
mechanisms by several bacterial physiological manifestations involving outer
membrane permeabilization, intracellular potassium ion efflux, intracellular nucleotide
leakage and alteration of bacterial cell morphology. The combination effects of
isolated antibacterial compounds from E. hirta with clinically established antibacterial agent, cefepime were evaluated via checkerboard broth microdilution method. The
crude methanol extract of E. hirta obtained the highest percentage yield (12.5 %) with
total phenolic content of 213.4 ± 0.75 mg GAE/g extract and total flavonoid contents
of 62.3 ± 1.01 mg CE/g extract. This study observed remarkable antibacterial activity
of the crude methanol extract against the resistant clinical isolate of Pseudomonas
aeruginosa with minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) values of 0.063 mg/mL and 0.125 mg/mL. Further fractionation
and isolation of the crude methanol extract produced three antibacterial compounds
which were identified as caffeic acid (CA), epicatechin 3-gallate (ECG) and quercitrin.
ECG had displayed the most promising anti-infective activity against P. aeruginosa
with similar potency as positive control, cefepime with MIC and MBC values of 16
μg/mL and 31 μg/mL respectively. CA was less active than ECG, at two fold lower
MIC and MBC values followed by quercitrin which showed the same efficacy as the
crude methanol extract. ECG is being isolated from E. hirta for the first time in this
study while CA and quercitrin are reported for the first time as having antibacterial
activity against P. aeruginosa. Study on the mechanism of action showed both ECG
and CA possessed bactericidal effect whereas quercitrin exerted bacteriostatic effect
against P. aeruginosa. ECG and CA were capable in disrupting the outer membrane
and cytoplasmic membrane of P. aeruginosa cell by enhancing the cell membrane
permeability. Scanning electron micrographs showed ECG and CA each caused
apparent cell membrane damages and leakages of cytoplasmic contents had eventually
led to cell death. In an attempt to further maximize antibacterial potency, combinations
of cefepime with ECG, CA and quercitrin respectively were tested individually against
P. aeruginosa isolate. Cefepime combined with ECG had produced synergistic effect with average FIC index of 0.24. However, combination of cefepime and CA showed
additive interaction while combination of cefepime and quercitrin displayed
indifference effect. To conclude, antibacterial constituents isolated from the
ethnomedicinal plant E. hirta, possessed remarkable anti-infective effect against
resistant P. aeruginosa pathogen. These bioactive molecules of clinical value await
further development as a new chemotherapeutic agent.
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Pharmacy